presence of mycoplasma using a standard PCR kit (EZ-PCR Kit, Gene flow (41106313-001)). On average outgrowths appeared within 14 days, with a small fraction of samples failing to produce outgrowths (12% of cases). Failures were due to contamination (0.5%) or lack of observed outgrowths after 30 days (11%). Approximately 30 days post dissection, when the fibroblasts had reached confluence, the culture was trypsinized and passaged into a 25 cm2 tissue culture flask. When 80-90% confluent, the fibroblasts were further passaged into a 75 cm2 flask. Cells were then expanded to confluency in 225 cm2 flasks (at a split ratio of 1:3) and either cryopreserved at 1-2 million cells per vial in FBS and 10% DMSO or seeded immediately for reprogramming.
